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MicroRNAs from the mediterranea: A model system for stem biology

DASARADHI PALAKODETI,1 MAGDA SMIELEWSKA,1 and BRENTON R. GRAVELEY Department of and , University of Connecticut Health Center, Farmington, Connecticut 06030-3301, USA

ABSTRACT MicroRNAs (miRNAs) are ;22-nt RNA molecules that typically bind to the 39 untranslated regions of target mRNAs and function to either induce mRNA degradation or repress translation. miRNAs have been shown to play important roles in the function of stem cells and cell lineage decisions in a variety of organisms, including humans. are bilaterally symmetric metazoans that have the unique ability to completely regenerate lost tissues or organs. This regenerative capacity is facilitated by a population of stem cells known as . Planarians are therefore an excellent model system for studying many aspects of biology. Here we report the cloning and initial characterization of 71 miRNAs from the planarian . While several of the S. mediterranea miRNAs are members of miRNA families identified in other , we also identified a number of planarian-specific miRNAs. This work lays the foundation for functional studies aimed at addressing the role of these miRNAs in , cell lineage decisions, and basic stem cell biology. Keywords: microRNA; Schmidtea mediterranea; planarians; stem cells

INTRODUCTION to other metazoans has not clearly been established. Planarians are bilaterally symmetric metazoans and are Planarians are best known for their amazing regenerative members of the phylum platyhelminthes (Newmark and capacity—even a fragment as small as 1/279th the size of the Sa´nchez Alvarado 2002). Early studies classified planarians original individual has the capacity to regenerate into an as acoelomates and, thus, placed them more distantly entire (Newmark and Sa´nchez Alvarado 2002). The related to vertebrates than insects or (Adoutte key to the regenerative prowess of these creatures are et al. 1999, 2000). In contrast, molecular analyses based on neoblasts, a population of totipotent cells distributed ribosomal RNA sequences have placed planarians in the throughout the organism (Newmark and Sa´nchez Alvarado lophotrochozoan clade of protostomes (Adoutte et al. 1999, 2002). Upon injury, the neoblasts migrate to the wound 2000). This classification suggests that planarians are site, divide, and their progeny eventually differentiate to actually more closely related to vertebrates than insects or replace the missing structures. Neoblasts also function in nematodes. In fact, of the planarian ESTs that are homol- homeostasis by serving to replace cells lost during normal ogous with other GenBank entries, 64% encode proteins cellular turnover. Thus, neoblasts are the planarian equiv- that are more similar to vertebrate than to inverte- alent of stem cells, making these remarkable creatures an brate genes (Sa´nchez Alvarado et al. 2002). However, excellent model system for studying stem cell biology a more recent analysis of metazoan phylogeny that com- (Sa´nchez Alvarado 2006). pared a common set of 50 genes from each organism Planarians are also extraordinarily interesting from an analyzed concluded that platyhelminthes are more similar evolutionary perspective because their precise relationship to nematodes than vertebrates or insects (Rokas et al. 2005). Soon, the genome sequence of the planarian Schmidtea mediterranea will be completed and this should 1These authors contributed equally to this work. Reprint requests to: Brenton R. Graveley, Department of Genetics and provide a clearer picture of the precise relationship of Developmental Biology, University of Connecticut Health Center, 263 planarians to other metazoans. Farmington Avenue, Farmington, CT 06030-3301, USA; e-mail: graveley@ MicroRNAs (miRNAs) are z18–25-nt long noncoding neuron.uchc.edu; fax: (860) 679-8345. Article published online ahead of print. Article and publication date are RNA molecules that play an important role in regulating at http://www.rnajournal.org/cgi/doi/10.1261/rna.117206. expression by binding to mRNAs and silencing the

1640 RNA (2006), 12:1640–1649. Published by Cold Spring Harbor Laboratory Press. Copyright Copyright Ó 2006 RNA Society.

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Planarian miRNAs

expression of the encoded proteins (Valencia-Sanchez tified in S. mediterranea, and one of these, SMEDWI-2, is et al. 2006). miRNAs are synthesized as large primary required for regeneration and homeostasis (Reddien et al. miRNA (pri-miRNA) transcripts that contain the mature 2005b). Though this observation does not directly impli- miRNAs embedded within distinct stem–loop structures. cate the miRNA pathway, it does suggest that small RNAs These stem–loops are sequentially processed by the RNa- play an important role in the control of homeostasis and seIII enzymes Drosha and Dicer (Kim 2005) and the regeneration in planarians. Thus, to fully understand how mature miRNAs are assembled into a miRNA Induced homeostasis and regeneration are regulated in planarians, it Silencing Complex (miRISC) containing a member of is important to identify planarian miRNAs and the target the Argonaute family of proteins. miRISC then associates mRNAs they regulate. with and silences target mRNAs (Valencia-Sanchez et al. As a first step toward investigating the role of miRNAs in 2006). regeneration, we report the cloning and characterization of Though the first miRNAs were identified genetically (Lee 71 miRNAs from the planarian S. mediterranea Impor- et al. 1993; Reinhart et al. 2000), hundreds of plant and tantly, this is the first lophotrochozoan organism from animal miRNAs have now been identified through molec- which miRNAs have been reported. Though many of the S. ular cloning and/or bioinformatic approaches (Lagos- mediterranea miRNAs belong to previously characterized Quintana et al. 2001; Lau et al. 2001; Lee and Ambros miRNA families in other metazoans, several are unique to 2001; Aravin et al. 2003; Lagos-Quintana et al. 2003; Lai et planarians. Interestingly, the majority of the conserved al. 2003; Lim et al. 2003; Jones-Rhoades and Bartel 2004; miRNAs are more similar to insect miRNAs than to Bentwich et al. 2005; Cummins et al. 2006). In , miRNAs from either nematodes or vertebrates. This work miRNAs function by imperfectly base-pairing with the 39 now makes it possible to begin studying the roles of untranslated regions of target mRNAs where they either miRNAs in controlling regeneration, cell lineage decisions, induce mRNA degradation (Yekta et al. 2004; Bagga et al. and stem cell function in planarians. 2005; Jing et al. 2005; Lim et al. 2005) or inhibit translation of the mRNA (Humphreys et al. 2005; Pillai et al. 2005; RESULTS AND DISCUSSION Petersen et al. 2006). It is thought that there are thousands of mRNAs that are subject to miRNA regulation in animals To begin to study the role of microRNAs in controlling (Brennecke et al. 2005; Krek et al. 2005; Lewis et al. 2005; regeneration, stem cell function, and cell lineage decisions Xie et al. 2005), though relatively few miRNA:mRNA pairs in planarians, we set out to identify microRNAs from have been experimentally validated to date. In cases where S. mediterranea. Our initial approach was to search the raw miRNA-mediated gene regulation has been validated, it is sequence reads of the unassembled S. mediterranea genome clear that miRNAs play criticial roles in many aspects of for sequences that are at least 90% identical to known development and cell lineage decisions (Ambros 2004; He human, Drosophila or miRNAs. and Hannon 2004). To cite just a few examples, miRNAs After identifying highly similar sequences, they were ana- have been shown to regulate developmental timing (Lee et lyzed with Mfold 3.2 (Zuker 2003) to determine whether al. 1993; Reinhart et al. 2000), cell proliferation (Brennecke the sequence surrounding the putative miRNAs could fold et al. 2003), left–right asymmetry in neural development into a stem–loop structure characteristic of pre-miRNAs. (Johnston and Hobert 2003), HOX gene expression This approach yielded only 10 potential miRNAs. We (Naguibneva et al. 2006), dendritic spine development therefore resorted to established cloning procedures (Lau (Schratt et al. 2006), and early embryogenesis (Giraldez et et al. 2001). Briefly, 18–26-nt RNAs were isolated from al. 2006). miRNAs also play important roles in stem cell both sexual and asexual strains of S. mediterranea, and biology. For example, a number of miRNAs that are specialized linkers were ligated to the 59 and 39 ends. These specifically expressed in human and mouse embryonic RNAs were reverse-transcribed, amplified by PCR, concat- stem cells have been described (Houbaviy et al. 2003; Suh enated, cloned, and the sequence of over 2200 individual et al. 2004). Moreover, miRNAs have been shown to inserts was obtained. control the differentiation of hematopoietic stem cells in To determine whether these sequences represent miR- mice (Chen et al. 2004) and germline stem cells in NAs, we first removed all sequences that were <18 nt (328 Drosophila (Forstemann et al. 2005; Hatfield et al. 2005). sequences) or >25 nt (five sequences) and subsequently Given the precision by which cellular proliferation and analyzed the remaining sequences to identify and remove differentiation must be coordinately controlled during contaminating rRNA, tRNA, or sequences that perfectly regeneration, and the fact that in other organisms miRNAs matched spliced ESTs (421 sequences). The remaining have been implicated in the control of stem cell function sequences were then used to search the raw sequence reads and cell lineage decisions, it is likely that miRNAs will play of the unassembled S. mediterranea genome, and those that a key role in regulating regeneration in planarians. Con- did not perfectly match the genome (685 sequences) were sistent with this hypothesis, two PIWI domain-containing discarded. Due to the fact that miRNAs display heteroge- proteins related to the Argonaute family have been iden- neity at their ends, the remaining 820 sequences were

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assembled into groups of related sequences that differed the context of the uncertainty regarding the phylogenetic slightly only at their 59 or 39 ends. Though heterogeneity relationship of planarians to other metazoans. was observed, only six instances of 59 end heterogeneity was To further validate these candidate miRNAs, the expres- observed—the remaining 30 cases involved only 39 end sion of 39 miRNAs was examined in both sexual and heterogeneity as expected, if these are authentic miRNAs. asexual strains of S. mediterranea, as well as in a second For each sequence we extracted 100 nt of genomic sequence planarian tigrina, by Northern blot experiments. flanking either side of the potential miRNA and tested Twenty representative northerns are shown in Figure 3. The whether stem–loop structures characteristic of known pre- expression of 33 of the miRNAs tested was detected in miRNAs could be formed by using Mfold 3.2 (Zuker 2003). S. mediterranea and all of these except for lin-4c were also This led to a nonredundant set of 81 potential miRNAs detected in D. tigrina. For miR-71c and miR-278, multiple represented by 773 individual sequences or 34% of all bands were detected on the Northern blots. This is sequences we identified (Supplemental Fig. S1; Supplemen- consistent with the cloning of multiple forms of these tary material can be found at http://genetics.uchc.edu/ miRNAs (Table 1). Six of the miRNAs were undetectable by graveley/supplemental/palakodetisupp.pdf). Importantly, Northern blotting. Three of these, miR-10*, miR-190b*, and of the 10 potential miRNAs identified by BLAST searching miR-277b*, are miRNA* strands and are therefore not with the human, Drosophila or C. elegans miRNAs, all but expected to be detected in this manner. One planarian- two, miR-8 and miR-13, were present in these clones. Given specific miRNA, miR-749, was cloned four times and may the high degree of validation of the predicted miRNAs, we be expressed at levels undetectable by Northern blotting. included miR-8 and miR-13 in our collection for a total of Two other miRNAs that were negative by northerns, miR-A 83 potential miRNAs (Tables 1, 2). and miR-B, are also planarian-specific miRNAs but were Previous studies have shown that, in addition to the cloned only once. These two miRNAs, along with five other mature miRNA, miRNA* strands, the complement of the planarian-specific miRNAs that were cloned only once and miRNA derived from Dicer cleavage of the pre-mRNA can not tested by northerns are considered as potential miRNAs occasionally be cloned (Lim et al. 2003). To determine and will require additional validation before being pro- whether any of the sequences we obtained corresponded to moted to being official miRNAs (Table 2). While it is miRNA* strands, we compared the sequence of each pre- possible that these sequences do not represent actual miRNA stem–loop with all others. This analysis revealed miRNAs, it is also possible that they are authentic miRNAs that 12 of the putative miRNA sequences actually corre- that are simply expressed at low levels. Regardless, these sponded to miRNA* strands (Table 1). Thus, we have results demonstrate that the majority of the miRNAs we identified a total of 71 potential miRNA genes from identified are not only expressed, but are also conserved in S. mediterranea (Supplemental Fig. S2; Supplemental other planarians. Although we did observe differences in materials are available online at http://genetics.uchc.edu/ the number of miRNAs cloned from the sexual and asexual graveley/supplemental/palakodetisupp.pdf). strains (Table 1), we did not observe any significant We next determined whether any of the potential difference in expression between the two strains by North- miRNAs we identified were similar to miRNAs in other ern blot (Fig. 3). This discrepancy could be reflective of the organisms by searching miRBase (Griffiths-Jones et al. overall quality of the miRNA libraries and/or cross hybrid- 2006) with the mature sequence of each putative miRNA. ization of the probes to highly similar miRNAs. Nonethe- We considered miRNA pairs to be members of a family if less, these experiments clearly demonstrate that most, if not two criteria were met. First, the seed regions, typically all, of these small RNAs represent authentic miRNAs. nucleotides 2–7 of the miRNA that serve to anchor the Although we identified four members of the let-7 family, miRNA to the mRNA target (Lewis et al. 2005), had to be we did not clone any miRNAs that perfectly matched the identical. Second, the mature miRNAs had to be located on sequence of the canonical let-7 (let-7a), which is 100% the same side of the pre-miRNA stem–loops. Using these identical in all metazoans examined so far (Pasquinelli et al. criteria, we found that 54 of the cloned miRNAs are 2000). Moreover, even BLAST searches of the S. mediterra- members of other metazoan miRNA families (Fig. 1), while nea genome, which has been sequenced to 83 coverage, did 17 appear to be unique to S. mediterranea. The conserved not identify any sequence that perfectly matched that of miRNA families include the lin-4 (Lee et al. 1993), let-7 let-7. Thus, it appears that S. mediterranea may not contain (Reinhart et al. 2000), and bantam (Brennecke et al. 2003). the canonical let-7 gene. Interestingly, only 14 of the miRNA families are conserved As is the case in other organisms, the miRNAs we in vertebrates, insects, and roundworms (Fig. 2). Surpris- identified frequently occur in clusters. Our ability to ingly, z80% of the conserved miRNAs have family mem- thoroughly assess the distribution of miRNAs throughout bers in insects, while only z50% have family members in the genome was limited by the fact that the S. mediterranea nematodes and vertebrates, despite the fact that more genome has yet to be assembled. Nonetheless, we examined miRNAs have been isolated from vertebrates and nemat- whether any of the contigs containing the miRNAs over- odes than from insects (Fig. 2). This finding is interesting in lapped with one another. This allowed us to identify seven

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TABLE 1. MicroRNAs from Schmidtea mediterranea

miRNA gene Sequence Lengtha Sexualb Asexualb Fold-back arm Conservationc Northernd

bantam-a UGAGAUCACUAUGAAAGCUGG 21–22 17 33 39 IY bantam-b UGAGAUCACUGCGAAAGCUGAU 21–22 0 9 39 IY* bantam-c UGAGAUCAUUAUGAAAGCUUUU 22 0 1 39 IY* let-7a UGAGGUAGAAUGUUGGAUGACU 21–22 2 11 59 V,I,N Y let-7b UGAGGUAGAUUGUUGGAUGACU 21–22 2 6 59 V,I,N Y* let-7b* CCAUUCAACUAUCUGUCUUCUC 22 1 0 39 N/A N/D let-7c UGAGGUAGUGACUCAAAAGGUU 22 1 12 59 V,I,N N/D lin-4a UCCCUGAGACCUUCGACUGUGU 20–22 7 61 59 V,I,N Y lin-4b UCCCUGAGACCAUUGACUGCAU 20–22 7 30 59 V,I,N Y lin-4b* GUAGUUUUUGGUAUCAGGAU 20 0 3 39 N/A N/D lin-4c UCCCUGAGAUCAUAAUAUGCCU 22 3 1 59 V,I,N Y miR-1a UGGAAUGUCGAGAAAUAUGCAU 12,22 3 5 39 V,I,N Y miR-1b UGGAAUGUCGUGAAUUAUGGUC 20,22,24 32 148 39 V,I,N Y miR-1c UGGAAUGUUGUGAAUAGUGUC 21 5 9 39 V,I,N Y miR-2a-1 UAUCACAGCCCCGCUUGGAACGCU 20–24 4 5 39 I,N Y miR-2a-2 UAUCACAGCCCCGCUUGGAACGCU 20–24 4 5 39 I,N Y miR-2a-2* GUUCUACGGUGUUGUGAUAU 20 0 1 59 N/A Y miR-2b UCACAGCCAAUUUUGAUGAGAU 19,22 0 4 39 I,N Y* miR-2b* UCAUCAUUGUUGGUUGUCAG 20 0 1 59 N/A N/D miR-2c UCACAGCCAAAACUGAUGAUCU 20–22 2 6 39 I,N Y miR-2d UCACAGCCAAAUUUGAUGUCC 18,21,22 48 26 39 I,N Y miR-7a UGGAAGACUAUUGAUUUAGUUGA 23 0 1 59 V,I N/D miR-7b UGGAAGACUGUCGAUUUCGUUGU 23 1 4 59 V,I N/D miR-7c UGGAAGACUGAUGAUUUGCUGA 22 0 4 59 V,I N/D miR-8 UAAUACUGUCAGGUAACGAUGCC 23 39 V,I Y miR-10 AACCCUGUAGAUCCGAGUUUGA 22 4 7 59 V,I Y miR-10* UCGAAUCUUCAAGGUGAA 19,22 0 1 39 N/A N miR-12 UGAGUAUUCUAUCAGGAGUCGA 22 0 1 59 I N/D miR-13 UAUCACAGUCAUGCUAAAGAGC 22 39 I N/D miR-31a AGGCAAGAUGUUGGCAUAACUGA 20–24 0 5 59 V,I,N Y miR-31b AGGCAAGAUGCUGGCAUAGCUGA 23 0 1 59 V,I,N Y* miR-36 UCACCGGGUAGACAUUCAUUA 21 1 0 39 N N/D miR-36* UGAUGCAUGCUACUUGGUUU 20 0 1 59 N/A N/D miR-61 UGACUAGAAAGUUCACUUACUGU 21,23 0 5 39 I,N N/D miR-67 UCACAACCUCCAUGAACGAGGGU 23 1 0 39 I,N N/D miR-71a-1 UGAAAGACACGGGUAGUGAGAU 21,22 1 23 59 NY miR-71a-2 UGAAAGACACGGGUAGUGAGAU 21,22,24 1 24 59 NY miR-71b UGAAAGACACAGGUAGUGGGAC 20–23 1 21 59 NY miR-71b* UCCUUCUAAUGUGUUUUUCG 20 0 1 39 N/A N/D miR-71c UGAAAGACAUGGGUAGUGAGAU 20–22 5 13 59 NY miR-79 GUAAAGCUAAAUUACCAAAGUGC 23,25 1 7 39 I,N N/D miR-87a UGAGCAAAGUUUCAAGUGUA 20 0 1 39 I,N N/D miR-87b GUGAGCAAAGCUUCAAAUGAG 21 0 1 39 I,N N/D miR-92 GAUUGCACUAGUUAAUUAUC 20 0 1 39 IY miR-124a UAAGGCACGCGGUGAAUGCUU 21 0 1 39 V,I,N Y* miR-124b UAAGGCACGCGGUGAAUGCUGA 20,22 0 3 39 V,I,N Y* miR-124c UAAGGCACGCGGUGAAUGCCA 19–22 3 19 39 V,I,N Y miR-124c* GCGCUCACCUCGUGACCUUUGU 21,22 1 2 59 N/A N/D miR-133 UUGGUCCCCAUCAACCAGCA 20 0 1 39 V,I N/D miR-184 GACGGAGGUUUGCUAAGGAA 19,20 0 2 39 V,I N/D miR-190a AGAUAUGUUUGGUUAAUUGGUGA 23 0 1 59 V N/D miR-190a* ACCACUGACCGAGCAUAUCCA 19–22 7 8 39 N/A Y miR-190b UGAUAUGUUUGGUUUAUUGGUGA 23 0 1 59 V N/D miR-190b* ACCAUUAGCCUAAUGUAUCGUGUA 20,24 2 0 39 N/A N miR-219 UGAUUGUCCAUACGCAGUUCUCA 21–23 1 4 59 V,I N/D miR-277a UAAAUGCACUAUCGGAUAUGAC 22 1 0 39 IY miR-277b AAAAUGCAUUAUCUGGCCAAGA 22 0 1 39 IY* miR-277b* UUGAUCAGAAAUGCAGCUUC 20 0 1 59 N/A N miR-277c UAAAUGCAUUAUCUGGUAUGAU 22,23 0 3 39 IY* miR-277d UAAAUGCAUUUAUCUGGCCAAG 22 1 0 39 IY (Continued)

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TABLE 1. Continued

miRNA gene Sequence Lengtha Sexualb Asexualb Fold-back arm Conservationc Northernd

miR-278 UCGGUGGGAGUAACAUUCGA 19–21 6 26 39 IY miR-281 UGUCAUGGAUAUGCUCUUCAU 19,21 1 1 39 I,N Y miR-281* UGAAGAGCUAUUCAUGAGGU 20 1 0 59 N/A Y miR-745 UGCUGCCUGGUUAAGAGCUGUGU 23 2 4 39 VY miR-746 UAGCACCAGGGUAUAUCGGGAU 22 0 2 39 V N/D miR-747 UAAUCUCAUCUGGUAAUUGAAGU 23 0 1 59 V N/D miR-748 UGGACGGAAGUGUAAUGAG 19 0 1 39 N/A Y miR-749 GCUGGGAUGAGCCUCGGUGGU 21 0 4 59 N/A N miR-750 UCAGAUCUAACUCUUCCAGUUCU 23 1 1 39 N/A Y miR-751 CAUGUUUGAAUGGCCAUGACA 20–21 1 2 39 N/A N/D miR-752 AGUCAGCAUUGGUGGUUU 18 0 1 39 N/A N/D miR-753 GAGCUUGGAUUGUGAUCUCA 20 0 1 59 N/A N/D miR-754 GUUGCUUGGGGUUAUUACUA 20 0 1 59 N/A N/D miR-755 UGGAGCUAUUGUAUUUCACC 18,20 1 1 59 N/A Y miR-756 CGAUAUGUGGUAAUUUGGAUGA 22 5 0 59 N/A Y

aVariations in length are due to 59 and 39 heterogeneity observed during miRNA cloning. The sequence of the most frequently cloned isoform for each miRNA is shown. bThe number of times each miRNA was cloned from the sexual and asexual strains is indicated. cOrganisms in which homologous miRNAs were identified are indicated. V, vertebrate; I, insect; N, ; N/A, no homolog identified. dThe results of Northern blots are shown. Y, positive northern result; Y*, sequence of miRNA is similar enough to another miRNA that was detected by Northern blotting in which cross-hybridization is possible; N, negative northern result; N/D, Northern blot not performed.

clusters that together account for 19 of the miRNAs or clusters, it is likely that these miRNAs arose by gene miRNA* strands we identified. The largest cluster contains duplication events followed by sequence divergence. For three stem–loop structures encoding the cloned miRNAs all of these clusters, it is likely that the linked miRNAs are miR-71b, miR-71b*, miR-2d, miR-752, as well as a fourth expressed as a single pri-miRNA. stem–loop containing one of the predicted miRNAs, miR-13 The collection of S. mediterranea miRNAs reported here (Fig. 4). We identified three additional clusters that each provides the foundation to begin exploring the miRNA- contain, one miR-2 family member and one miR-71 family mediated gene regulatory networks that are involved in member. Each of these clusters adopts similar secondary controlling homeostasis and regeneration. Determining the structures, yet displays little sequence similarity to one expression patterns and predicting potential target mRNAs another aside from the mature miRNAs (Fig. S3A–D). for each of these miRNAs should point toward potential In fact, two of these clusters, miR-71a-1/miR-2a-1 and miRNA:mRNA pairs that could play a role in regulating miR-71a-2/miR-2a-2, contain identical miRNAs. We also regeneration. Although thousands of S. mediterranea ESTs identified two clusters that contain two members of the have been cloned (Sa´nchez Alvarado et al. 2002; Zayas et al. miR-277 family each (Fig. S3E,F): One cluster contains 2005), accurate and robust miRNA target prediction must miR-277a and miR-277b, while the other contains miR-277c await assembly of the genome so that a complete, high and miR-277d. For both the miR-71/miR-2 and miR-277 quality 39 UTR data set can be compiled. Nonetheless, once

TABLE 2. Potential MicroRNAs from Schmidtea mediterranea

miRNA gene Sequence Length Sexuala Asexuala Fold-back arm Conservationb Northernc

miR-A CACUCCUGCCCAAUCCCUUUC 21 0 1 59 N/A N miR-B GGUAGGCAUAGUCUGUUGGUUAC 23 0 1 59 N/A N miR-C GUCCCACCUGAGAUGCCA 18 0 1 39 N/A N/D miR-D GGGGUGCAAGCUCUUGAUUGAAGUC 25 0 1 39 N/A N/D miR-Ea UGACCACUGGUGCGAUCCUGC 21 0 1 39 N/A N/D miR-Eb UGACCAUGGACGCGACCCUAC 21 0 1 39 N/A N/D miR-F CCUUACGGCCCCUAAACCUUAUU 23 0 1 39 N/A N/D miR-G GAUUGCACUCAAUUAUGGUCAGA 23 1 0 39 N/A N/D

aThe number of times each miRNA was cloned from the sexual and asexual strains is indicated. bThese miRNAs are not similar to miRNAs from other organisms. cThe results of Northern blots are shown. N, negative northern result; N/D, Northern blot not performed.

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FIGURE 1. MicroRNA families. Alignments of S. mediterranea miRNAs with family members from human, Drosophila,orC. elegans miRNAs were performed with ClustalW. In some instances, only one representative from each family is shown.

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acrylamide gel, and the ligated products excised and eluted as before. The RNAs were then ligated to 100 mM59 linker oligo (59-ATCGTrArGrGrCrArCrCrUrGrArArA-39) (Intergrated DNA Technology) in a reaction containing 0.4 mM ATP, 1 U/mLT4 RNA ligase for 6 h at room temperature. The ligation mixture was again resolved on a 10% denaturing polyacrylamide gel and the ligated products excised and eluted as before. The ligated RNA molecules were converted to cDNA with SuperScriptII (Invitro- gen) and the Reverse Transcription (RT) primer (59-ATTGATGG FIGURE 2. Relationships between metazoan miRNAs. A Venn TGCCTACAG-39). Subsequently, the products were amplified by diagram depicting the number of S. mediterranea miRNAs that have PCR using the RT primer and the 59 primer (59-ATCGTAGGCA family members in vertebrates, insects, or nematodes. CCTGAAA-39) using the following protocol: 96°C, 1 min, 26 cycles of 96°C 10 sec, 50°C 1 min, 72°C 20 sec, followed by a 3-min incubation at 72°C. The PCR products were phenol-extracted, potential targets are identified, they can be validated ethanol-precipitated, and digested with BanI (20 U/mL). The through a combination of RNA interference (Newmark digested products were concatemerized by incubation with T4 DNA ligase (NEB) and the reaction mixture was resolved on a 2% et al. 2003; Reddien et al. 2005a), transgenic (Gonzalez- low-melting agarose gel. Concatemers ranging from 300 to 600 bp Estevez et al. 2003), antisense (Leaman et al. 2005), and were eluted from the gel, tailed with Taq DNA polymerase, and microarray approaches. Such studies should provide insight cloned into pPCR-2.1-TOPO (Invitrogen). Colony PCR was per- into how miRNA-mediated gene regulation contributes to formed to identify clones containing inserts >2300–600 bp in size the control of homeostasis and regeneration in planarians. and those clones were sequenced. This will also increase our understanding of how stem cells are regulated in planarians, which may very well be similar Bioinformatic analysis in humans. The sequences of individual inserts between 18 and 25 nt were used in local BLAST searches to screen databases of S. mediterranea MATERIALS AND METHODS

Planarian culturing Asexual and sexual strains of S. mediterranea were kind gifts from A. Sa´nchez Alvarado and P. Newmark. S. mediterranea was maintained at room temperature (21–22°C) in ddH2O supple- mented with 1.6 mM NaCl, 1.0 mM CaCl2, 1.0 mM MgSO4, 0.1 mM MgCl2, 0.1 mM KCl, 1.2 mM NaHCO3, and fed homoge- nized beef liver. Dugesia tigrina were obtained from Carolina Biological Supply and were maintained in Poland Spring water at room temperature. All animals were starved for one week prior to any experiments.

Small RNA cloning Small RNA isolation and cloning from S. mediterranea were done essentially as described previously (Lau et al. 2001). Total RNA was separately isolated from sexual and asexual animals using Trizol as described by the manufacturer (Invitrogen). Trace amounts of 32P-59-end-labeled markers of 18 (AGCGUGUAGGG AUCCAAA) and 24 (GGCCAACGUUCUCAACAAUAGUGA) nt were added to each RNA sample and then separated on a 15% denaturing polyacrylamide gel. RNAs ranging from 18 to 24 nt were excised from the gel and eluted overnight in 0.4 M NaCl at 4°C. The eluted RNA was precipitated with two volumes of ethanol in the presence of 1 mg/mL of glycogen as carrier. The FIGURE 3. Northern blot analysis of miRNA expression. Fifty purified RNA was ligated to the modban linker (AMP-59p-59p-CT micrograms of total RNA isolated from both sexual and asexual GTAGGCACCATCAATdi-deoxyC-39) (Integrated DNA Technol- strains of S. mediterranea and another planarian, Dugesia tigrina, were resolved on 15% denaturing polyacrylamide gels and transferred to ogy) with 1 U/mL T4 RNA ligase (NEB) for 2 h at room temp- membranes. Ethidium bromide staining of the gels indicated that erature in a reaction mixture contatining 50 mM HEPES pH 8.3, equal amounts of RNA were loaded into each well. The blots were 32 5 mM MgCl2, 3.3 mM DTT, 10 mg/mL BSA, and 8.3% glycerol. probed with P, 59-end-labeled oligonucleotides complementary to The ligation mixture was separated on a 10% denaturing poly- the miRNAs indicated.

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resulting contigs containing the miRNA clusters were reexamined with MFold.

Northern blots Total RNA was extracted using TRIzol (Invitrogen) according to the manufac- turer’s protocol. Fifty micrograms of total RNA was loaded in each lane and resolved on 15% denaturing polyacrylamide gel. The gels were stained with ethidium bromide to confirm equal loading of the samples and then transferred onto a GeneScreen Plus membrane (Perkin Elmer), UV cross-linked, and baked for an hour at 80°C. Oligonucle- otide probes complementary to the miRNAs were 32P-end-labeled with T4 kinase and hybridized overnight at 42°C in 7% SDS and 0.2 M Na2PO4, pH 7.0. The blots were washed in 23 SSC, 0.1% SDS at room temperature, and visualized with a Cyclone Phosphoimager (Perkin Elmer).

ACKNOWLEDGMENTS We thank Alejandro Sa´nchez Alvarado and Phil Newmark for providing the sexual and asexual S. mediterranea animals. We thank Amy Pasquinelli, Erik Sontheimer, Asis Das, Gene Yeo, and members of the Graveley lab for comments on the manuscript and discussions. This work was generously supported by the Raymond and Beverly Sackler Fund for the Arts and Sciences to FIGURE 4. A S. mediterranea miRNA cluster. The predicted secondary structure of a pre- miRNA containing miR-71b, miR-71b*, miR-2d, miR-752, and a predicted miRNA, miR-13. B.R.G. The sequence of the mature miRNAs is shown in red and the miRNA* strands that were identified are shown in blue. Received April 18, 2006; accepted May 26, 2006.

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MicroRNAs from the Planarian Schmidtea mediterranea: A model system for stem cell biology

Dasaradhi Palakodeti, Magda Smielewska and Brenton R. Graveley

RNA 2006 12: 1640-1649

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